High Resolution Capillary Electrophoresis ("HRCE") in small bore capillaries (such as less than or to 75 .mu.) was first demonstrated by Jorgenson and Lukacs, and has proven useful as an efficient method for the separation of small solutes. J. Chromatog., 218 (1981), page 209; Anal. Chem., 53 (1981), page 1298.
Attractive factors for electrophoretic separations by capillary zone electrophoresis are the small sample sizes, little or no sample pretreatment, and the potential for quantification and recovery of biologically active samples. The separation process is strongly influenced by an electroosmosis effect, generally described as the flow of a liquid in contact with a solid surface under the influence of a tangentially applied electric field. Electroosmotic flow ("EOF") and differences in electrophoretic mobilities combine to provide a spatial separation of constituents of the sample solution at the outlet end of the capillary tube.
Electrophoretic migration is the movement of charged constituents in response to an electric field. A positively charged molecule will migrate towards the cathode, while a negatively charged molecule will migrate towards the anode. The net movement of the charged species in the separation chamber is then governed by the vector sum of the electrophoretic mobility ("EPM") and the electroosmotic flow ("EOF"), given by: EQU .mu. total=EOF+.mu. EPM
It is apparent from this expression that if the EOF is greater than the electrophoretic mobility of a species migrating against the flow, then the net migration of the species will be in the direction of the EOF. Consequently, electroosmotic flow is a high efficiency mass transport means for moving neutral and oppositely charged constituents of a sample past a single point of detection.
For open tubular electrophoresis, it is necessary to have control over the EOF in order to optimize resolution and analysis time. Many experimental conditions, such as surface modification, field strength, buffer pH, ionic strength and species and organic modifiers such as solvents or surfactants, may alter EOF. Currently, in order to attempt to optimize separation parameters, practitioners have had systematically to vary the different conditions in attempts to optimize separations.
After a decade of intense interest and development in open tubular capillary electrophoresis as an analytical technique, fused silica (usually externally coated with polyamide) has been the capillary tubing of choice. As a high energy surface, its greatest single weakness is its affinity for a wide variety of solutes of interest. The untoward solute/surface interactions are a major source of loss of efficiency and reproducibility in this evolving separation technique. Numerous attempts at surface deactivation have been reported, ranging from dynamic deactivation using additives in the electrophoresis solution to specific chemical modification of the silica surface. All deactivation techniques ultimately have an impact on the EOF.
U.S. Pat. No. 4,680,201, issued 1987, inventor Hjerten, describes a method for preparing a thin wall capillary tube for electrophoretic separations by use of a bifunctional compound in which one group (usually a terminal--SiX.sub.3 group where X=ethoxy, methoxy or chloride) reacts with the glass wall and the other (usually an olefin group) does so with a monomer taking part in a polymerization process. This process is said to result in a wall-bonded, polymer-coated capillary useful for open tubular electrophoresis.
U.S. Pat. No. 4,931,328, inventor Swedberg, issued Jun. 5, 1990, describes a modified capillary tube that has an interfacial layer covalently bonded to the inner wall of the capillary tube. The interfacial layer is effective to reduce interactions between the inner wall and protein solutes, and includes a hydratable amphoteric phase. This amphoteric phase has a determinable isoelectric point and permits electroosmotic flow control by selection of solution pH.
U.S. Pat. No. 5,006,313, issued Apr. 9, 1991, inventor Swedberg, describes capillary tubes with a reduced interaction phase coated along the bore for reducing interactions of protein solutes with the surface. When this interfacial layer is about 4 to about 6 molecular layers thick, then it has been found that electroosmotic flow is reasonably high in use for capillary zone electrophoresis.
Lee et al., Anal. Chem., 62 (1990), pages 1550-1552, reported a technique in which capillary electroosmosis with bare fused silica had an external electric field applied to modify the zeta potential at the aqueous/inner capillary interface. That is, this technique factorially couples externally applied potential with the potential across the buffer solution inside the capillary. This electric potential gradient across the capillary wall vectorially sums with the polarity and magnitude of the charged double ion layer at the surface/liquid interface of the interior surface of the capillary, which is the so called "zeta potential". The resultant direction and flow rate of electroosmosis is dependent upon the combination of the external transverse field and the zeta potential polarity and the zeta potential magnitude.
Despite the advances, current HRCE column technology generally suffers from two major disadvantages: lack of ability to vary the EOF component when flow control is retained, independent of the electrophoretic mobility of solute species of interest; and, undesirable solute/surface interactions. The former disadvantage greatly limits the ability to optimize separation conditions and analysis time and the latter degrades the reproducibility and efficiency of the separation technique.